The invention relates generally to the field of test sample preparation and analysis. More particularly, the subject invention relates to a method and apparatus for automatically purifying a test sample and enhancing the sensitivity of the sample analysis.
Flow cytometry is a well known technique for qualitatively and quantitatively analyzing a large number of individual cells for a specific cellular marker in a rapid manner. In a typical application, a fluorescent molecular probe that selectively binds to a predetermined cell marker, such as a fluorochrome-conjugated antibody that specifically binds an intracellular or cell surface antigen, is added to a cell sample to be analyzed so that the probe can bind or xe2x80x9cstainxe2x80x9d the cells within the sample that express the predetermined cell marker. The sample is then placed in flow cytometer and illuminated with a light source to enable the fluorescence associated with each cell in the sample to be quantified. The magnitude of fluorescence emitted from a particular cell correlates with the quantity of cell marker on or in that particular cell. By extrapolating this fluorescence data, the relative quantity of specific phenotypic markers expressed by cells in a sample can be rapidly and accurately determined. For an overview of flow cytometric analysis see, xe2x80x9cFlow Cytometry and Sorting,xe2x80x9d Myron R. Melamed, Tore Lindmo, and Mortimer L. Mendelsohn, eds., New York:Wiley-Liss, Inc., (3rd ed., 1995); Shapiro, H. M., xe2x80x9cPractical Flow Cytometry,xe2x80x9d New York:Wiley-Liss, Inc., (2nd ed., 1990).
Sample preparation for flow cytometric analysis is typically performed in a non-automated fashion, wherein a saturating concentration of a cell marker-specific probe is added to a cell sample by manual pipetting, and the mixture is then incubated for a period of time sufficient to allow the probe to bind the cell marker of interest. For analyses where red blood cells might cause interference (e.g., immuno-phenotyping leukocytes), the red blood cells can be removed from the sample using an agent that specifically lyses erythrocytes (for example, a hypotonic solution, ammonium chloride or carboxylic acid). Traditionally, to remove interfering unbound probe from the cell sample prior to flow cytometric analysis, the mixture is washed by adding excess buffer to the mixture, centrifuging the mixture to separate the cells from the buffer, removing the buffer containing the unbound probe, and resuspending the cells in fresh buffer. The washing procedure can be repeated multiple times to further remove any remaining unbound probe. This non-automated technique is advantageous in that it results in a relatively clean sample that contains few interferants (for example, unbound probe or cell debris) which might generate background noise or interference during the flow cytometric analysis. For many applications, however, this non-automated technique is relatively time-consuming, can result in significant cell loss due to one or more wash steps, and exposes the cells to the potentially deleterious effects (for example, activation of enzymatic processes, granule release, cell destruction, high gravity forces produced by centrifugation, etc.).
While the foregoing technique is acceptable for infrequent analyses involving a small number of samples, it is less suitable for protocols involving repeated analyses of a large number of samples. A more automated procedure is generally preferred when flow cytometric analysis is employed for clinical diagnostics, high-throughput screening, or the like. For example, in a typical clinical assay where leukocytes are immunophenotyped using flow cytometry, a sample of whole blood is placed into an apparatus that automatically processes the sample prior to analysis. One such apparatus is the COULTER(copyright) TQ-Prep(trademark) Workstation system manufactured by Beckman Coulter, Inc. (Miami, Fla.). After adding a probe to the sample, this apparatus uses computer-controlled devices to automatically add an agent that lyses erythrocytes in the sample and a cell fixing agent (for example, paraformaldehyde). The prepared sample can then be analyzed using a flow cytometer without further processing. This automated technique is advantageous in that samples of whole blood can be prepared for analysis quickly and efficiently.
A drawback of this lysing technique can be encountered in applications requiring a high degree of sensitivity. In such applications, in the absence of a washing step, the automated technique does not remove interferants, such as unbound probe or debris from the lysed erythrocytes from the sample. The high background signal caused by the fluorescence from the unbound probe, non-specific probe binding, and/or autofluorescence from the cells and debris can obscure results generated from the analysis.
Where a fluorescently-labeled antibody is used to analyze a cell sample for a marker present in low quantities, the absence of a washing step can result in high background fluorescence caused by the unbound antibody present in the sample. Thus, if too many unbound fluorescent antibody molecules are present in the sample, the flow cytometer can not distinguish the signal emitted from the antibody-bound cells from the xe2x80x9cnoisexe2x80x9d generated by the unbound antibody. That is, the xe2x80x9cnoisexe2x80x9d in the sample overwhelms the xe2x80x9csignalxe2x80x9d emanating from the cells of interest. To avoid this, the signal to noise ratio in the sample can be improved by removing the interferants by manually washing. An example of manual washing comprises centrifuging the sample to pellet the cells, decanting the interferants contained in the supernatant, and resuspending the cells in fresh buffer. As described above for the non-automated technique, this manual washing is disadvantageous because it is time consuming, causes cell damage, and can result in significant cell loss.
A need therefore exists for an apparatus and method for quickly and efficiently removing interferants from a cell sample prior to analysis. In addition, the apparatus and method should minimize the risk of exposure to infectious blood because of operator handling of the blood cell sample. An apparatus that performs the foregoing method with only negligible cell loss, and does not expose cells to high gravitational forces or cell packing caused by centrifugation would be especially advantageous.
Similarly, there exists a need for an apparatus and method for quickly and efficiently removing interferants from a test sample prior to analysis. In addition, the apparatus and method should minimize the risk of exposure to test sample because of operator handling of the test sample. An apparatus that performs the foregoing method with only negligible loss of the composition of interest in the test sample, and does not expose test sample to high gravitational forces caused by centrifugation would be especially advantageous.
It has been discovered that filters, such as microporous hollow fiber membranes, can be utilized in sample preparation devices to quickly and efficiently remove interferants from a test sample comprising a mixture of a composition of interest and interferants. More specifically, it has been found that the use of a hollow fiber membrane having a plurality of pores with a mean diameter less than the diameter of the composition of interest can be utilized to remove interferants from a test sample containing the composition of interest and interferants to improve the signal-to-noise ratio in an assay of the composition of interest. Application of vacuum to the hollow fiber membrane permits interferants to be removed from a test sample within a lumen of the filter with little or no damage to the composition of interest. As the composition of interest does not pass through pores of the membrane, compared with conventional continuous filtration devices, clogging of the filter is less frequent, and the composition of interest is exposed to less deleterious forces. Filters within the invention can be installed in a processing apparatus such that a test sample can be washed and analyzed automatically.
Accordingly the invention features an apparatus for automatically removing interferants from a test sample containing a composition of interest and interferants. The apparatus includes a vacuum source; a filtration device comprising an impermeable housing that forms an extramembrane chamber wherein said chamber contains a filter that selective separates a composition of interest from a mixture of the composition of interest and interferants, and wherein said housing contains at least two ports, preferably more than three ports, and wherein at least one port is connected by a conduit to the vacuum source; a conduit from one of said ports in said housing which is adapted to aspirate the mixture of the composition of interest and interferants from a container into the filtration device by said vacuum source; and a conduit from one of said ports in said housing which fluidly connects to a buffer reservoir, which provides a means for buffer to enter into said filtration device and exit through one of said ports. The apparatus further includes a conduit, which is fluidly connected to an analyzer that analyzes the composition of interest by suitable means, such as an electrical measurement and optical measurement.
In a preferred embodiment, the apparatus for automatically removing interferants from a test sample containing a mixture of a composition of interest and interferants includes recovery of the composition of interest through the same conduit which is adapted to aspirate the test sample from the test sample container.
The filter of the apparatus preferably includes a microporous hollow fiber membrane having a plurality of pores sized such that the composition of interest is prevented from passing through the hollow fiber membrane. For example, the pores can have a mean diameter of between about 0.1 and 5.0 microns. In preferred versions of the apparatus, the microporous hollow fiber membrane is fashioned into at least one tube defining a lumen, the tube having a first port providing a first opening in the tube, and a second port providing a second opening in the tube. In this preferred embodiment, the conduit can be fluidly connected to the at least one lumen via the first port such that the test sample can be moved from the test sample container through the first port into the at least one lumen. The second port can be fluidly connected to a buffer reservoir containing a buffer and also fluidly connected to a detergent solution reservoir containing a detergent solution. The means for recovering the cells from the filtration device can include a fluid pump that can be in fluid communication with a buffer reservoir suitable for housing a buffer so that the fluid pump can cause the buffer to flow from the buffer reservoir into the filtration device. In variations, the fluid pump can also cause the buffer to flow from the filtration device into the at least one conduit.
In another aspect of the apparatus of the invention, the invention provides an automated method for removing interferants from a mixture of a composition of interest and interferants comprising applying a vacuum force to a first container containing a mixture of a composition of interest and interferants to cause the mixture containing the composition of interest and interferants to contact a filter; applying a force to said mixture in contact with the filter to selective separate the composition of interest from the mixture of the composition of interest and interferants; and recovering the composition of interest from the filter. Preferably the force used to enable the filter to selectively separate the composition of interest from the mixture is a vacuum force. In another aspect, the apparatus of the invention can include a computer controller for controlling the pumps and valves.
The invention also features an automated method of preparing a body fluid for analysis comprising adding at least one an analyte specific bead that reacts with a body fluid to form a test sample mixture containing an analyte specific bead complex and interferants; automatically removing interferants from said test sample mixture to yield a washed analyte specific bead complex; and analyzing the washed analyte specific bead complex to determine a characteristic of the body fluid.
The invention further features a method for purification of a proteinaceous material from a mixture of the proteinaceous material and interferants comprising supplying a first end of a hollow fiber filter with a mixture of a proteinaceous material having a molecular weight between approximately 50,000 and 1,000,000 and interferants having a molecular weight that is less than 50% of the molecular weight of the proteinaceous material to a first end of a hollow fiber filter; applying a pressure force to a lumen of the hollow fiber filter to cause the interferants in the mixture to pass through the membrane of the hollow fiber filter; adding buffer or other fluid which does not react with the proteinaceous material to further cause the interferants to pass through the membrane of the hollow fiber filter; and recovering the proteinaceous material from a second end of the hollow fiber filter, said second end being disposed at an opposite end of the hollow fiber filter from the first end. The proteinaceous material is selected from the group consisting of antibodies, activated antibodies, fluorescent labels, activated fluorescent labels, and conjugated antibody fluorescent label.
In addition, the invention even further features a method for a method for purification of a biological macromolecule from a mixture of the biological macromolecule and interferants comprising supplying a first end of a hollow fiber filter with a mixture of a biological macromolecule having a molecular weight between approximately 20,000 and 2,000,000 and interferants having a molecular weight that is less than 50% of the molecular weight of the biological macromolecule to a first end of a hollow fiber filter; applying a pressure force to a lumen of the hollow fiber filter to cause the interferants in the mixture to pass through the membrane of the hollow fiber filter; adding buffer or other fluid which does not react with the biological macromolecule to further cause the interferants to pass through the membrane of the hollow fiber filter; and recovering the biological macromolecule from a second end of the hollow fiber filter, said second end being disposed at an opposite end of the hollow fiber filter from the first end. The biological macromolecule is selected from the group consisting of nucleic acids and complex carbohydrates.
The methods further includes analyzing the composition of interest, the proteinaceous material or the biological macromolecule by suitable means, such as an electrical measurement and optical measurement.